Non-equilibrium, fuel-driven reaction cycles serve as model systems of the intricate reaction networks of life. Rich and dynamic behavior is observed when reaction cycles regulate assembly processes, such as phase separation. However, it remains unclear how the interplay between multiple reaction cycles affects the success of emergent assemblies. To tackle this question, we created a library of molecules that compete for a common fuel that transiently activates products. Often, the competition for fuel implies that a competitor decreases the lifetime of these products. However, in cases where the transient competitor product can phase-separate, such a competitor can increase the survival time of one product. Moreover, in the presence of oscillatory fueling, the same mechanism reduces variations in the product concentration while the concentration variations of the competitor product are enhanced. Like a parasite, the product benefits from the protection of the host against deactivation and increases its robustness against fuel variations at the expense of the robustness of the host. Such a parasitic behavior in multiple fuel-driven reaction cycles represents a lifelike trait, paving the way for the bottom-up design of synthetic life. Non-equilibrium, fuel-driven reaction cycles serve as model systems of the intricate reaction networks of life. Thus, a population of transient product molecules emerges whose properties are regulated kinetically. In recent years, examples of such reaction cycles have been introduced that regulate the ability of molecules to assemble or phase-separate, resulting in dynamic structures like colloids,9,10 fibers,10–16 supramolecular polymers,17–19 oil-based droplets,20,21 coacervate-based droplets,22,23 vesicles,24–26 micelles,27 particle clusters,28–31 macrocycles32 and DNA-based nanostructures.33,34 Due to the transient nature of these building blocks, these assemblies are endowed with properties typically absent at thermodynamic equilibrium. For example, fibrils that spontaneously self-divide,23 temporary hydrogels,10,35–37 or oil-based emulsions of which ripening is accelerated.2 Moreover, the theory on active emulsions suggests that droplets can self-divide.38,39 More recently, examples of assemblies were observed that exert feedback over their chemical reaction cycle.11,27,40,41 The underlying mechanisms can result in exciting behavior like the spontaneous emergence of switches between the morphologies or the ability of molecules to persist while others decay.9,20 All these developments in the field are incremental steps towards the synthesis of life, and a living system essentially represents a complex non-equilibrium assembly of molecules that is regulated by chemical reaction cycles.42–44 However, in living systems, a vast number of reaction cycles operate simultaneously and interact in intricate networks through feedback mechanisms. While such systems show interesting and complex emergent properties in a close-to-equilibrium context,45–50 the behavior of multiple reaction cycles in fuel-driven synthetic systems has been underexplored. In particular, competition and feedback are expected to lead to interesting emergent behavior in such systems. For example, oil droplets showed that selection and inhibition can occur in systems competing for a common fuel.51In chemically fueled systems, the propensity of molecules to form assemblies is regulated by a chemical reaction cycle. In the cycle, a precursor is activated at the expense of a chemical fuel.In this work, we show an unexpected behavior in phase-separated emulsions that are regulated by chemical reaction cycles and compete for a fuel. Counterintuitively, the lifetime of a transient product can be vastly prolonged even when resources have to be shared. The underlying mechanism is based on co-phase separation which protects products against deactivation, and it shows similarity to how a parasite benefits from the presence of a host.52We used a chemical reaction cycle that is driven by the hydration of the condensing agent EDC carbodiimide). In the activation reaction, the fuel condenses a succinate derivative into its corresponding anhydride product . In the via confocal microscopy  and 2-hexen-1-ylsuccinate . The hydrolysis rate can then be calculated by r = kdcout, where kd is the hydrolysis rate constant. Since both kd and cout are constant, the effective hydrolysis rate is constant leading to a linear decay of the total anhydride product concentration when all fuel is consumed. Indeed, when using this equation in a theoretical kinetic model, we can accurately predict the concentration of fuel, succinate derivative and anhydride product for all three chemical reaction cycles . When we fueled 50 mM precursor with 100 mM EDC, we found that the precursor is immediately converted to roughly 25 mM of the product and, after the depletion of the fuel, degraded rapidly with a first-order decay within 24 minutes . Next, wversuscout of competitor 2 which means that droplets dissolve below this threshold. Moreover, the threshold value is not in the tailing regime of the exponential decay of the anhydrides allowing to capture the effects of phase separation on product lifetime -2-buten-1-ylsuccinic anhydride (product) and (E/Z)-2-hexenyl-1-ylsuccinic anhydride (product of competitor 2) from TCI chemicals. Succinic acid (competitor 1), succinic anhydride (product of competitor 1), Nile Red, 1-ethyl-3-(3 dimethyl-aminopropyl)carbodiimide hydrochloride (EDC), trifluoroacetic acid (TFA) and 2-(N-morpholino)ethane sulfonic acid (MES buffer) were purchased from Sigma-Aldrich. All chemicals were used without any further purification unless otherwise indicated. High performance liquid chromatography (HPLC) grade acetonitrile (ACN) was purchased from VWR.We purchased (E/Z)-2-Buten-1-yl-succinic anhydride (product) and (E/Z)-2-hexenyl-1-ylsuccinic anhydride (product of competitor 2) were dispersed in 30 mL MQ water and stirred for 3 days. Subsequently, the reaction mixture was freeze-dried, and the corresponding succinates (precursor and competitor 2) were stored at −20 °C until further usage. to monitor the concentration profiles of each reactant of the chemical reaction network. We prepared 1.0 mL samples according to the sample preparation protocol described above into a screw cap HPLC vial. Samples were injected directly from the HPLC vial without any further dilution. We injected 25 μL for the detection of the succinates and anhydrides and 1 μL for the detection of EDC. We used a UV/Vis detector at 220 nm for the detection. A linear gradient of MQ water : ACN with 0.1% TFA was used to separate the compounds. We used a linear gradient from 98 : 2 to 2 : 98 in 10 minutes followed by 2 minutes at 2 : 98 for the separation. The column was equilibrated for 2 minutes after each gradient. We performed calibration curves of the compounds in triplicates. Calibration values and retention times are given in ESI Tables S1 and S2.†We imaged the droplets using a Leica SP8 confocal microscope with a 63× oil immersion objective. Samples were prepared as described above but with 0.1 μM Nile Red added before EDC addition. We added 5 μL sample to a silicon grease reservoir on a PEG-coated glass slide covered with a 12 mm diameter coverslip. The samples were excited with a 543 nm laser and imaged at 580–700 nm.We prepared 5 mL samples as described above to guarantee a sufficient droplet phase volume after centrifugation. After the depletion of the fuel, the reaction mixture was centrifuged at 4 °C for 3 minutes (rpm = 5000). We used an Eppendorf pipette to take a 1 μL sample of the droplet pellet which we diluted in 200 μL ACN in a HPLC screw cap vial. We analyzed the ratio of product to product of competitor 2 with HPLC.We prepared 1 mL samples as described above in 1.5 mL Eppendorf reaction vessels. After 16 minutes, the samples were centrifuged at 25 °C for 1 minute (rpm = 13 500). We directly analyzed the concentrations of the corresponding anhydrides in the supernatant of the sample with HPLC (see ESI Tables S4 and S5nI = ntotal − nII and their molecular volumes, respectively (see ESI Tables S3, S5–S7VI.We calculated the corresponding anhydride concentrations inside the droplets using the relation J. B. and P. S. S. designed the experiments. P. S. S. and T. H. carried out the experiment. S. L., J. J. and C. A. W. worked out the theory. S. L. and J. J. performed the numerical studies and the fitting to the experimental data. P. S. S., S. L., J. B. and C. A. W. wrote the manuscript. All authors have given approval to the final version of the manuscript.There are no conflicts to declare.SC-012-D1SC01106E-s001


All eyes showed a reduction in retinal thickness, with the central macular thickness being 470 ± 151 μm at baseline and 360 ± 144 μm at the last follow-up (p = 0.001). Intra-retinal fluid was present at baseline in 12 eyes (63%) and in three eyes (16%) at the last follow-up (p = 0.065). Sub-retinal fluid was present at baseline in 17 eyes (89%) and at the last follow-up in three eyes . Pigment epithelium detachment was apparent in the 16 eyes (84%) at baseline and was still present in 14 eyes . One adverse event of intraocular inflammation was reported. In conclusion, our short-term experience showed that brolucizumab was highly effective in restoring the anatomy and in stabilizing the visual acuity of eyes with nAMD. Its safety profile should be evaluated carefully and needs further investigations.To report the early efficacy and safety outcomes of treatment with intravitreal injections of brolucizumab (IVT-B) in patients presenting neovascular age-related macular degeneration (nAMD) in a tertiary clinical setting. A retrospective case series of patients that received IVT-B with a minimum of two injections performed and at least 4 weeks of follow-up after last injection. Nineteen eyes of 19 patients were included. The number of IVT-B performed for the whole cohort was 58 injections; the mean number of IVT-B per patient was 3.0 ± 1.0 (range 2–6); the mean follow-up time was 14.4 ± 9.0 weeks. Mean baseline best-corrected visual acuity was 0.4 ± 0.4 logMAR and at the last follow-up was 0.4 ± 0.6 logMAR ( Brolucizumab, the newest agent approved in clinical use for the treatment of neovascular age-related macular degeneration, was designed by grafting the complementarity-determining regions of a novel anti-VEGF-A antibody onto a human single-chain antibody fragment. Lately, the American Society of Retinal Specialists highlighted some Adverse Drug Reactions in patients receiving brolucizumab. The present study reported short-term effective anatomical and functional outcomes of brolucizumab in a real-world practice, which confirm results from randomized clinical trials. These data are important for the ophthalmic community and the pharmacovigilance system, particularly for the safety profile. The safety profile of brolucizumab needs further investigations and signs of intraocular inflammation should be evaluated with careful patient monitoring.®, Novartis Pharma AG, Basel, Switzerland) is a low molecular weight (26 kDa versus 48 kDa of ranibizumab and 115 kDa of aflibercept), single-chain antibody fragment that targets all forms of VEGF-A with high affinity (Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss in people aged 55 years and older in western industrialized countries . Neovascaffinity . The resaffinity . Broluciaffinity ; so theraffinity . Clinicaaffinity , thus ouThis is a retrospective, observational, monocentric study at the Jules Gonin Eye Hospital. Clinical records of nAMD patients treated with IVT-B in routine clinical practice at our Institution from March to December 2020 were reviewed. Inclusion criteria were a diagnosis of nAMD with any type of choroidal neovascularization involving the foveal region, a minimum of two injections, including the loading dose, and 4-weeks follow-up following the last IVT-B. Exclusion criteria were the presence of macular diseases other than nAMD, and a history of intraocular inflammation. Treatment-naïve nAMD eyes as well as eyes already under treatment with anti-VEGF intravitreal injection for active choroidal neovascularization (CNV) secondary to nAMD were enrolled. For treatment-naïve eyes, the decision of treatment with IVT-B was proposed to the patient following an extensive discussion of the risks and benefits involved. For patients already receiving IVT injections, the decision of switching to brolucizumab was proposed by the retina specialist based on active exudation persisting after at least three IVTs of other anti-VEGF agents (ranibizumab or aflibercept). The first IVT-B was performed 1 month after the last ranibizumab and 2 months after the last aflibercept. The presence of refractory active exudation was assessed by a senior retina specialist (C.M.E) on the basis of OCT qualitative features suggestive of exudative disease activity following previously published guidelines . All patPrior to the first IVT-B, all patients underwent best-corrected visual acuity (BCVA) measurement using an Early Treatment Diabetic Retinopathy Study (ETDRS) chart, intraocular pressure (IOP) measurement, a complete slit lamp biomicroscopy examination, fundus ophthalmoscopy following pupil dilation with 1% tropicamide eye drops, and optical coherence tomography (OCT)  imaging.In the case of treatment-naïve nAMD eyes, fluorescein (FA) and indocyanine green (ICGA) angiography were also performed . Follow-up visits were monthly for the first 3 months, and bimonthly (q8) or every 3 months (q12) subsequently, accordingly to the treatment interval. At each follow-up visit, patients underwent BCVA and IOP measurements, slit-lamp anterior segment and fundus biomicroscopic examinations and OCT imaging.https://www.swissmedic.ch/swissmedic/en/home/about-us/publications/public-summary-swiss-par/public-summary-swisspar-beovu.html. Accessed 30 Nov 2020). An initial loading dose of three monthly IVT-B injections was scheduled. Safety evaluations were conducted at every visit, with special regard to signs of IOI. The symptoms of IOI were explained to each patient and they were advised to contact the clinic immediately, should any of these symptoms  appear during follow-up. Treatment intervals following the loading dose were determined by a senior retinal specialist (CME) based on clinical evaluation of the anatomical and functional response to the first three doses and a q8 or a q12 interval was adopted as necessary, in accordance with the registration trials  Working Group guidelines .Collected data included the demographic characteristics and ophthalmic history of the patients, the number of previous intravitreal injections, functional (BCVA) and anatomical parameters, and postoperative complications. For non-treatment-naïve eyes, data just prior to the first IVT-B was considered the baseline, and the subsequent data after brolucizumab injections were included in the analysis. Therefore, in this study we analyzed BCVA at baseline before the IVT-B and at the last follow-up. Central macular thickness (CMT) was measured in OCT volume scans in the central subfield of an Early Treatment of Diabetic Retinopathy Study grid, centered on the fovea. Centering to the fovea and retinal layer segmentation during OCT examination were manually controlled and adjusted if needed for fixation or segmentation misalignments. The presence of intra-retinal fluid (IRF), sub-retinal fluid (SRF), and pigment epithelial detachment (PED) were recorded in OCT B-scans at baseline and at the last follow-up visit by two independent retinal specialists (CME and AM). A qualitative grading as resolution, reduction or increase of IRF, SRF, and PED from baseline to last follow-up was also performed based on the OCT B-scans by two independent retinal specialists (CME and AM). In case of disagreement, a third retina specialist (FS) graded the scans.t-test with a 95% CI. Correlation for multiple testing was performed using the Bonferroni test. A two-tailed Fisher’s exact test was used to analyze the association between categorical variables with results statistically significant when associated with a p-value ≤ 0.05.Statistical analysis was performed using SPSS software . Data were analyzed with frequency and descriptive statistics for qualitative variables. The normality of data samples was assessed with the Kolmogorov–Smirnov test. Statistical comparison between baseline and the last follow-up data for continuous variables was performed using Student’s paired Nineteen eyes of 19 patients met the inclusion criteria and were included in the analysis. The baseline characteristics of the study population are summarized in The complete functional and anatomical parameters are summarized in p = 0.778, 95% CI = −0.15 to 0.11, post-hoc statistical power analysis 7.2%). Seven eyes (37%) gained at least one line of vision, nine eyes (47%) remained stable, and three eyes (16%) lost one line of vision.Mean BCVA at baseline was 0.4 ± 0.4 [range from −0.1 to 1.3] logMAR [Snellen equivalent 20/50 (20/16—20/400)], and at the last follow-up was 0.4 ± 0.6 [range from −0.1 to 2] logMAR . IRF was present in 12 eyes (63%) at baseline and in three eyes (16%) at the last follow-up. This change was not statistically significant (p = 0.065). SRF was present in 17 eyes (89%) at baseline and in three eyes (16%) at the last follow-up visit. This difference was statistically significant (p = 0.011). The OCT B-scan revealed the presence of a PED in 16 eyes (84%) at baseline and in 14 eyes (73%) at the last follow-up, with no statistical difference between the two time points (p = 0.811)  at baseline and 360 ± 144 µm (range 203–728) at last the follow-up (= 0.811) . At the = 0.811) , 2; PED 35–802 at= 0.811) . DiffereIn our sample, we found one ocular adverse event. An 85-year-old female who had previously received 82 injections of aflibercept and 12 injections of ranibizumab was switched to brolucizumab due to refractory SRF. The last ranibizumab injection was delivered 4 weeks prior to the first brolucizumab injection, and she received a total of two injections of brolucizumab. Three days after the second IVT-B, she presented with a mild decrease in visual acuity (from 20/50 right before the IVT-B to 20/63), anterior chamber inflammation and an intermediate uveitis classified as mild vitreitis following the Standardization of Uveitis Nomenclature (SUN) Working Group criteria (vitreous haze on dilated fundus microscopy with the details of the posterior pole slightly hazy).. FA and p = 0.011) at the last visit compared to baseline. The reduction magnitude of IRF and PED size was overall positive even though it did not reach statistical significance . None of the eyes showed a deterioration of their anatomical condition at baseline in terms of any of the study parameters.In the present study, we reported our short-term real-life clinical experience with using intravitreal brolucizumab for the treatment of nAMD patients. At last follow-up, all eyes in this cohort showed a decrease of the CMT with mostly a complete resolution of IRF and SRF and a stabilization of the BCVA. In the registration trials HAWK and HARRIER, brolucizumab showed a greater fluid resolution and a non-inferior visual function in comparison to aflibercept, which were maintained until week 96 . The resp = 0.778).This may be attributed to the fact that most of the eyes in our cohort presented a longstanding history of nAMD with chronic intra- and sub-retinal fluid accumulation that might limit the potential for visual recovery. Moreover, the mean number of previous anti-VEGF injections in the majority of the enrolled eyes was 32, with only four treatment-naïve eyes, while the HAWK and HARRIER trials included selected treatment-naïve eyes only, with a relatively good visual acuity (mean baseline BCVA of Snellen 20/63)  . Eyes win 20/63) . In addin 20/63) , which wn 20/63) .The presence of fluid is a hallmark of disease activity and therefore retinal fluid control is the aim of nAMD management. So far, brolucizumab has shown encouraging results in reducing the presence or amount of fluids in the different retinal compartments . In thiswww.brolucizumab.info). Although the pathogenesis of these AEs has not been established with sureness and is currently under investigation  presented baseline low vision and in 5 (26%) of them the presence of atrophic and/or fibrotic changes or amblyopia limited the potential for visual recovery. However, even in these eyes with chronic lesions, refractory to other anti-VEGF drugs, the good anatomical response to IV-B with complete resolution of fluid resulted in a stabilization of visual acuity and a subjective improvement.On the other side, we believe that these results represent indeed a strength of this study. In fact, the heterogeneous population enrolled, characterized by a wide spectrum of baseline clinical features and duration of the disease, represents a real-life situation that differs from the selected patients in clinical trials and therefore, it reflects the treatment outcomes of a real clinical practice. To our knowledge, this is one of only a few reports so far on real-life outcomes of intravitreal brolucizumab for the treatment of nAMD. For this reason, we believe that it is of interest to the ophthalmic community and the pharmacovigilance system. Of course, reports on larger cohorts and longer follow up are needed to support the preliminary real-life experiences of this treatment.In conclusion, in our early real-life experience, brolucizumab resulted highly effective in restoring the anatomy of eyes affected by nAMD and in stabilizing the visual acuity of both treatment-naïve eyes and of eyes refractory to other anti-VEGF intravitreal agents. Nevertheless, the safety profile of this new anti-VEGF treatment should be evaluated carefully and needs further investigations.


Motor neuron diseases such as spinal cord injuries and amyotrophic lateral sclerosis are known as the most common disorders worldwide. Using stem cells  is currently a potent medical approach for modulating the impact of neural damages and regeneration of spinal cord injuries. MicroRNAs (miRNA) are taken into account as principal regulators during differentiation. The miRNAs play a significant role in stem cell self-renewal and fate determination. There are few studies on how miRNAs regulate neural differentiation in stem cells. The purpose of this study is to explore miRNA profiles of CB-MSCs during differentiation into motor neuron-like cells. Human CB-MSCs were isolated and characterized using flow cytometry. Cell differentiation has been induced by combining retinoic acid (RA) and sonic hedgehog (Shh) in a two-step protocol for 14 days. Then, cell differentiation was confirmed by immunocytochemistry and flow cytometry. The miRNA was analyzed using Illumina/Solexa sequencing platform. In this regard, three libraries were prepared to investigate the effect of these two biological morphogens on the miRNA profile of the differentiating cells. These libraries were Control (non-treated CB-MSCs), Test 1 (RA + /Shh +), and Test 2 (RA-/Shh-). Quantitative RT-PCR was employed to verify miRNA expression. CB-MSCs were spindle-shaped in morphology, and they did not express hematopoietic markers. After differentiation, the cells expressed motor neuron markers  at the protein level after 14 days. The analysis of miRNA sequencing demonstrated a significant up-regulation of miR-9-5p and miR-324-5p in Test 1 (RA + /Shh +). Also, there is a considerable down-regulation of mir-137 and let-7b in Test 2 (RA-/Shh-). These results have been obtained by comparing them with the Control library. Indeed, they were responsible for neuron and motor neuron differentiation and suppression of proliferation in neural progenitor cells. Furthermore, significant up-regulation was detected in some novel microRNAs involved in cholinergic, JAK-STAT, and Hedgehog and MAPK signaling pathways. CB-MSCs are potent to express motor neuron markers. This procedure has been performed by developing a two-week protocol and employing Shh and RA. The miRNA profile analysis showed a significant up-regulation in the expression of some miRs involved in neuron differentiation and motor neuron maturation. MiR-9-5p and miR-324-5p were up-regulated at the early stage of differentiation. Also, miR-137 and miR-let-7b were downregulated in the absence of RA and Shh. Furthermore, several novel miRNAs involved in cholinergic, Hedgehog, MAPK, and JAK-STAT signaling pathways have been detected. However, further studies are still necessary to validate their functions during motor neuron generation and maturation. These regulators play a principal role in stem cell self-renewal and fate determination. In this regard, crucial tasks are defined for gene regulation. This procedure is performed by degrading messenger RNAs or inhibiting gene translation2.The miRNAs represent a group of non-coding RNAs that consist of 18–24 nucleotides regulating post-transcriptional gene expression3. Thus, the expression of a series of genes that control biological processes is modulated by one miRNA. It plays a crucial role in cellular processes such as differentiation and cell proliferation during normal development4. It is due to its capacity for coordinating the control of gene expression and the broad range of expression across different types of cells.Every miRNA has the potential to identify multiple target mRNAs5. For example, miR-9 transiently expression during motor neuron differentiation regulates the expression of FoxP1, and a HOX accessory factor coordinates motor neuron subtype identity and connectivity. In mice, miR-9 overexpression induces neuronal differentiation by inhibiting the nuclear TLX receptor15. A positive regulator of the Wnt signaling pathway16 is essential for neuronal progenitor self-renewal. Also, let-7b and miR-137 are principal regulators for neural stem cell proliferation. These are significantly up-regulated during neural differentiation. Also, they suppress the expression of TLX23.In recent years, different investigations have attempted to identify the link between miRNAs and neurogenesis. The results demonstrated that non-coding RNAs are crucial components to modulate transcriptional networks associated with neural development and function6. RA and Shh play a principal role in specifying motor neurons in the central nervous system7. According to in-vitro studies, the administrations of RA8 and Shh9 support the differentiation of Wharton's jelly, olfactory mucosa10, bone marrow, and chorion-derived mesenchymal stem cells into motor neuron-like cells11. It appears that RA and Shh regulate the expression of transcription factors, which are essential for specifying motor neurons in the spinal cord and a synergistic manner11.There are few protocols to induce stem cell-derived motor neurons. For example, transplantation of BDNF-overexpressing hUC-MSC-derived motor neurons could improve motor performance and prolong the survival of the amyotrophic lateral sclerosis (ALS) model in mice. In the developing neural tube, the generation of motor neurons along with the rostrocaudal axis is spatiotemporally orchestrated by a set of morphogens such as sonic hedgehog (Shh) and retinoic acid (RA)12. The study conducted by Hohjoh and Fukushima regarding the up-regulation of 19 miRNAs during neurogenesis in mouse and human cells after RA treatment indicated that their functions are conserved in mammalian species13. For example, they reported that miR-30214 and miR-124a exhibited the opposite expression patterns in response to RA treatment at the beginning of neural differentiation. Moreover, the suppression of some miRNAs  directs the differentiation of neuronal progenitors into motor neurons and decreases the interneuron population15.The downstream molecular interactions of RA and Shh signaling pathways are often unclear. However, it seems that post-transcriptional silencing carried out by miRNAs has a vital role in the spatiotemporal regulation of neuronal specification4. Despite the concerns of neuroscientists about the application of stem cells in neurogenesis, few studies have been performed on how miRNAs regulate neural differentiation in stem cells. Therefore, studies on miRNA profiles of differentiating stem cells are beneficial to identify the effect of potent miRNAs on neurogenesis.Many types of researches have been performed in the field of neurogenesis. However, there is little knowledge related to the role of miRNAs in the neurogenesis process. Indeed, these researches have failed to portray a dynamic profile of the miRNA expression at the time of neurogenesis16. The hUCB-MSCs are known as proper cell candidates for regenerating the lost cells in the central nervous system17. These cells have self-renewal features18, immunomodulatory properties, and neural differentiation capacities. This study is focused on differentiating human CB-MSCs into motor neuron-like cells and examining the miRNA profile of the motor neuron-like cells derived in the presence of RA and Shh. In this study, the surface markers of CD90, CD44, and CD73 expressed by hMSC and specific markers  were characterized as the negative markers for the MSCs19. In this study, the differentiation was induced by RA and Shh and applying the proposed two-step protocol. In this regard, the results revealed that the cells could express motor neuron markers  at the protein level. The miRNA sequencing showed a significant up-regulation in the expression of miR-9-5p and miR-324-5p at the early stage of the differentiation. This procedure occurred in the presence of RA and Shh (RA + /Shh + test group). The miR-137 and let-7b were down-regulated in Test 2 (RA-/Shh-). It was confirmed by comparing the achieved results with the Control library. They were all responsible for neuron and motor neuron differentiation and suppression of proliferation in neural progenitor cells, respectively. Also, we could detect some novel miRNAs involved in cholinergic, JAk-STAT, and Hedgehog and MAPK signaling pathways during differentiation. Further experiments are still essential to validate their expression and functions during motor neuron generation and maturation.Human umbilical cord blood (UCB) is a postpartum medical waste product. It is known as a rich source of stem cells. The UCB has several advantages over other sources of stem cells in regenerative medicine, including convenient access, a higher amount of pluripotent cells, higher toleration across the barrier, and a slighter prevalence of graft-versus-host disease (GVHD)20. In this study, we collected blood samples using a 50 ml sterile syringe that contained 10 µl of heparin. Then, the samples were moved to the lab and placed on an ice pack. Almost four hours after collecting samples, red blood cells have been removed by adding 1 ml of 10% Hydroxyethyl starch . Then, the supernatant was collected and washed with phosphate-buffered saline . Next, it was supplemented with 100 U/ml penicillin, 0.2% EDTA, 15% fetal bovine serum (FBS) , and 0.1 mg/ml streptomycin . After centrifugation over Ficoll-Hypaque  at a rate of 300 g for 15 min, mononuclear cells were achieved and cleansed in PBS. Afterward, a total of 5 × 103 cells/cm2 were cultured in 75 cm2 tissue culture flasks. These flasks contained DMEM-F12 with 100 μg/ml streptomycin, 10% FBS, and 100 U/ml penicillin . These were placed in a dampened chamber at 37 °C with 5% CO2. The medium was refreshed one week later. After observing the first colonies, the medium was changed every day. Afterward, at 60% confluence, new culture flasks were used to plate cells, and the medium was modulated every three days. The following experiments contained cells at passage three.After gaining informed consent from mothers, we gathered hUCB-MSCs from the umbilical vein of infants delivered by elective cesarean. Using cells for this study was endorsed by the Ethical Committee of Iran University of Medical Sciences. The hUCB-MSCs were isolated based on pre-defined protocols20. A total of 1 × 105 cells were seeded in every well from a 24-well culture plate and incubated at dampened chamber overnight. On the next day, the expansion medium was replaced by the medium used before induction. This medium contained 20% fetal bovine serum, DMEM-F12, 10 ng/ml of fibroblast growth factor , 250 mM of isobutylmethylxanthine (IBMX), 2% B27 , and 100 mM of β-mercaptoethanol. We retained the cells in a dampened chamber overnight. On the next day, they were treated with the induction medium that contained DMEM-F12 with 0.01 mM of all-trans RA , 100 ng/ml supplementation of Shh , and 0.2% B27  for a week . Then, the RA and Shh were removed at the end of the first week. Afterward, the medium was substituted with DMEM-F12 and supplemented with 100 ng/ml of brain-derived neurotrophic factor (BDNF)  and 0.2% B27 as surviving factor for one more week . A summary of the protocol is illustrated in flow chart.The hUCB-MSCs were induced into motor neuron-like cells based on the protocols defined in the previous step This flow chart is showing the study design and the experimental groups. Test 1 is the group of MSCs treated with RA/Shh collected for analysis at day 7. Test 2 is the same group of MSCs which was kept in the culture medium without RA/Shh for addition of 7 days. Fetal bovine serum (FBS), fibroblast growth factor (FGF), isobutylmethylxanthine (IBMX), surviving factors (B27 + BDNF) and β-mercaptoethanol (β-ME).20. In this regard, we incubated 1 × 105 cells with 10% goat serum in PBS at a temperature of 4 °C. After spending one hour, the serum was withdrawn, and the incubation of cells was performed either by phycoerythrin (PE)-conjugated monoclonal antibodies or fluorescein isothiocyanate (FITC) against human CD34, CD45, CD44, CD73, and CD90  at 4 °C for 40 min. All antibodies were diluted in PBS/0.1%BSA at the concentration of 1:200. Also, the control library was comprised of isotype-matched antibodies. The expression of antigens was detected using BD FACSCalibur. In this study, the data analysis was performed by FlowJo v7.6.1 software.Mesenchymal stem cells were characterized by mono-color cytofluorimetric analysis. This process was carried out based on the protocol defined by the authors in the previous studyn = 3) and the average of the expression level of each marker was quantified as mean ± standard error of mean. Also, the expressions of Islet-1, ChAT, and SMI-32 proteins were observed by Olympus DP70 fluorescent microscope after nuclei staining with DAPI . It was performed based on the mentioned immunostaining protocol10.Cytofluorimetric analysis has quantitatively been carried out to identify the expression of Islet-1, ChAT, and SMI-32 antigens at the end of the second week (T2 time point). At the end of the proposed two-week protocol, the differentiated cells were fixed in 4% paraformaldehyde for 4 h. The permeabilization analysis was performed by incubating the cells with 0.2% Triton X-100 in PBS  at room temperature. The cells were washed by PBS twice, and then they were incubated with 10% goat serum in PBS for 60 min at room temperature. Afterward, samples were incubated with primary antibodies (1:200) against human ChAT , SMI-32 , and Islet-1  at 4 °C. In the next step, the cells were washed by PBS twice. Then, we conducted incubation using the matching phycoerythrin (PE) or fluorescein isothiocyanate (FITC) conjugated secondary antibodies  for 45 min at 37 °C. The expression of antigens was detected by BD FACSCalibur (BD Biosciences) at the end of the defined two weeks protocol. In this context, positive expression was defined as a fluorescence level above 95% compared to the one measured by the corresponding isotype-matched control antibody. Data were analyzed by Flowjo v7.6.1 software. For quantification of the flow cytometry data, the expression level of each three markers; including ChAT, Islet-1, and SMI-32 was investigated in three different biological replicates , total cellular RNA was extracted using TRIzol reagent . After digestion of DNA, the purity of RNA was quantified. Complementary DNA (cDNA) was synthesized using RevertAid H Minus First Strand cDNA Synthesis Kit . Real-time PCR reactions were carried out using 7500 Real time PCR system . In each PCR reaction, X1 SYBR Green PCR Master Mix  was mixed with 12 ng of cDNA and the related primers , 250 mM of isobutylmethylxanthine, 2% B27 , and 100 mM of β-mercaptoethanol. The cells have been retained in a dampened chamber overnight. On the next day, these were treated with the induction medium for a week. It contained DMEM-F12 with 100 ng/ml supplementation of Shh , 0.01 mM of all-trans RA , and 0.2% B27 . The cells were collected and named as “RA+/Shh+ group” or Test 1. Afterward, the remaining cells have been kept in the medium for one more week. The medium was contained DMEM-F12 without RA and Shh and supplemented with survival factors, including 100 ng/ml of BDNF  and 0.2% B27. The cells were collected and named as “RA–/Shh– group” or Test 2. For sequencing, the sample size was ≥ 1 µg, the concentration was ≥ 50 ng/µl, and the sample volume was about 15 µl. These quantities were considered based on BGI's general sample requirement guideline. Similar quantities of total RNA from labeled control and two test groups (RA/Shh+ and RA–/Shh–) were pooled, and the sample has been run in the sequencer.In this experiment, three libraries have been provided, including “Control”, “RA/Shh+ and RA–/Shh–) were pooled. The purified gel products of the ligation have been converted into DNA. Then, the outcome was amplified by RT-PCR with 15 PCR cycles to create libraries. They were sequenced by Illumina/Solexa platform sequencer at Beijing Genomics Institute (BGI), Hong Kong.The RNA extraction kit has been used to extract total RNA from the cell groups . This procedure was accomplished based on the instructions provided by the manufacturer. After incubation, genomic DNA was withdrawn using RNase-Free DNase . This process was followed by an RNA Clean Purification Kit . This procedure was performed for 15 min at a temperature of 37 °C. Also, Agilent 2100 Bioanalyzer was utilized to assess the quality and integrity of RNAs. The purification of total RNA has been performed using the electrophoretic separation on a 15% denaturing polyacrylamide gel. This process was followed by excision and recovery of small RNA regions (15–30 nucleotide bands). Then, the sequences of proprietary adapters were ligated to the 5′- and 3′-termini of these small RNAs. Similar quantities of total RNA from labeled control and two test groups (RA/Shhhttp://www.sanger.ac.uk/Software/Rfam) and NCBI GenBank (http://www.ncbi.nih.gov/GenBank/) databases. The miRNA database (miRBase 20.0) has been utilized to detect preserved miRNAs. In this case, a maximum of two mismatches was allowed. The Mireap software (https://sourceforge.net/projects/mireap/) has been employed to detect novel miRNAs. Thus, the novel miRNAs were predicted by the rest of the unknown small RNAs (sRNAs). The principal criteria are adopted to screen the potential novel miRNAs. In this study, Mfold was utilized  to construct the stem-loop structures for putative pre-miRNAs. If a complete stem-loop structure was formed, the sRNA sequence would have landed at a stem arm together with other ensuing criteria. Thus, this sRNA was taken into account as a new miRNA.Extensive raw sequences are collected from high-throughput sequencing. Following the removal of impure sequences , we queried unique reads against non-coding RNAs such as rRNAs, tRNAs, small nucleolar RNAs (snoRNAs), and small nuclear RNAs (snRNAs) in the Rfam (http://www.geneontology.org/) was employed for functional annotation in the candidate target gene of miRNAs. The Kyoto Encyclopedia of Gene and Genome (KEGG) pathway database (http://www.kegg.jp/kegg/) has been employed to detect remarkably enriched signal transduction or metabolic pathways in target gene candidates and compare them with the whole reference gene background. The pathway analysis shed further light on the biological functions of target genes.In this section, the miRanda program has been considered to predict target genes for the identified miRNAs. The candidate targets were BLASTn hits that had less than four mismatches. Also, the NCBI database and target sequences were utilized to predict the functions of the candidate targets by BLASTX. The enrichment analysis of gene ontology (GO)  and the TREEVIEW software.If miRNA has no read in the library, its normalized read count is randomly set to 0.01 for further computations. If the expression level of a miRNA was below 20 reads, the difference analysis is abandoned. The hierarchical cluster analysis was conducted by CLUSTER 3.0 . This procedure has been carried out using the RNA extraction kit . Genomic DNA was removed using RNase-Free DNase , which has been later followed by an RNA Clean Purification Kit . Also, the reverse transcription and qRT-PCR reactions were performed by DNase-treated RNA samples. A total volume of 20 μL has been considered, and then the reactions contained 5 µM of each primer, 2.5 ng of cDNA, and 10 μL of SYBR Green  considering the cycling profile at 95 °C for 30 s, which was accompanied with 40 cycles at 95 °C and 60 °C for 5 and 20 s, respectively. QRT-PCR was performed in a 7500 real-time PCR system . A triplicate processing of each sample was conducted, and 18SrRNA was adopted as an internal control. The qRT-PCR primers are provided in supplementary n = 3 in each group) were statistically assessed by the nonparametric Mann–Whitney test using SPSS ver. 16. P values of < 0.05 were considered significant.Data were expressed as the mean ± standard error of mean, and differences between studied groups , Tehran, Iran.Cell morphology assessment and Flow cytometric analysis have been performed for characterizing the isolated cells. The isolated MSCs taken from human umbilical cord blood had spindle-like morphology at passage three Fig. a.Figure The isolated cells were distinguished based on a panel of positive and negative selection markers. They could express CD44, CD73, and CD90 markers on their surfaces at passage three. However, they were negative for hematopoietic antigens such as CD45 and CD34 Fig. b.n = 3) revealed that 40.427 ± 7.078%, 47.557 ± 12.475%, and 25.137 ± 3.872% of the treated cells could express Islet-1, ChAT and SMI-32 at second week of induction, respectively . The slight expression of Hb-9 was also detected. No significant difference was observed in the expression of the candid genes between T1 and T2  at T1 and T2 . These low-expression miRNAs were not used for further expression analysis. The read counts were different between 328 known miRNAs. In these three libraries, the miRNAs belonged to 56 conserved families. Overall, the numbers of 466, 452, and 400 miRNA families were identified in the Control, Test 1, and Test 2 groups, respectively  were eliminated. Thus, 77 potential novel miRNAs were predicted in the three libraries Table . AccordiIn this section, the putative targets were predicted for the conserved and novel miRNAs by the miRanda program. Then, the functions of the conserved and novel miRNAs have been predicted. The Gene Ontology (GO) analysis has been performed to evaluate the possible functions of the miRNA target genes. The detected miRNAs targets have been predicted based on the human genome sequence. Many target positions have been predicted for the known conserved miRNAs in the sequencing libraries. A total of 621, 624, and 562 potential target genes were identified for the conserved miRNAs in the Control, Test 1 (RA + /Shh +), and Test 2 (RA-/Shh-) groups. Also, 16, 39, and 38 potential target genes have been identified for the novel miRNAs in the Control, Test 1 (RA + /Shh +), and Test 2 (RA-/Shh-) groups, respectively. This information is provided in Table The data between groups have been analyzed to find the miRNAs. The results showed significant up or down-regulation in comparison with the control group. The reads of the known and novel miRNA sequences were compared in the three libraries to find the differentially regulated miRNAs in the Control, Test 1 (RA + /Shh +), and Test 2 (RA-/Shh-) groups. In these libraries, the expression levels of the conserved Fig. a and newOverall, 111, 128, and 239 conserved miRNAs had different expressions in the Control versus Test 1 (RA + /Shh +), Control versus Test 2 (RA-/Shh-), and Test 1 (RA + /Shh +) versus Test 2 (RA-/Shh-), respectively. A comparison between the miRNA expression profiles in Test 1 group (RA/Shh +) and the Control group showed that ten miRNAs were down-regulated. For example, hsa-miR-449c-5p, hsa-miR-1249-3p, hsa-mir-9-5p, and mir-324 were up-regulated. While hsa-miR-335-3p and hsa-miR-335-5p were significantly down-regulated.A comparison between the miRNA expression profiles in Test 2 (RA/Shh-) and the Control groups demonstrated that 106 miRNAs were down-regulated. For example, miR-let7b, miR-137, hsa-miR-149-3p, hsa-miR-3940-3p, and hsa-miR-6511-5p were down-regulated, whereas hsa-miR-619-5p and hsa-miR-3687 were significantly up-regulated. A comparison between the expression profiles in Test 1 group and Test 2 group revealed that 230 miRNAs  were down-regulated in Test 2. Also, nine miRNAs  were up-regulated.A total of 32, 35, and 41 novel miRNAs showed different expression levels in the Control versus Test 1, Control versus Test 2, and Test 1 versus Test 2, respectively. A comparison between the miRNA expression profiles in Test 1 and Control groups showed that 27 miRNAs were up-regulated. For example, novel-mir-17, 18, and 20 were up-regulated, whereas novel-miR-1 and novel-miR-2 were significantly down-regulated.A comparison between the miRNA expression profiles in Test 2 and Control groups suggested that 30 miRNAs were up-regulated. For example, novel-miR-2, 3, and 6 were down-regulated, whereas novel-miR-18, 19, and 20 were significantly up-regulated.Also, the expression profile of the Test 1 group has been compared with the Test 2 group. The results revealed that 21 miRNAs were down-regulated, while 20 others were up-regulated. The miRNAs such as novel-miR-1, novel-miR-52, and novel-miR-53 were up-regulated Table .Table 6DThe real-time quantitative PCR (qRT-PCR) analysis has been completed to verify and measure the differential expression levels of miRNAs. The proposed known and novel miRNAs exhibited significant expression differences in the three libraries. The expression levels of the six conserved miRNAs and six new miRNAs in the Control, Test 1, and Test 2 groups were validated and measured by the qRT-PCR technique. All randomly chosen miRNAs were identified by high-throughput sequencing in the Control, Test 1, and Test 2 groups. According to the qRT-PCR analysis, all tested miRNAs have been identified in the groups Fig. . These mSince motor neuron-related disorders and diseases are dramatically common worldwide, the importance of stem cell-derived motor neurons as an in vitro model used for drug screening in neuromuscular disorders is not evitable. Many types of researches have been performed to provide motor neurons via various stem cells, including MSCs and iPSCs. These approaches have been developed for use in neural injury modeling and drug screening. Although both miRNA-based differentiation of hiPSCs and mesenchymal stem cells into neural lineages have been reported, there is still little knowledge regarding the role of miRNAs in regulating motor neuron differentiation.The purpose of this study was to explore miRNA profiles of human CB-MSCs during differentiation into motor neurons in the presence of two morphogens, naming RA and Shh. These morphogens were responsible for motor neuron generation. After differentiation induction, the cells expressed motor neuron-related markers  at the level of mRNA and protein. This procedure was performed using immunocytochemistry and flow cytometry. In this study, three small RNA libraries have been provided to investigate the miRNA profile of differentiating cells. These libraries were derived through the following groups: (1) The cells treated by RA and Shh: Test1 (RA + /Shh +), (2) The cells after removing of RA and Shh from the medium: Test2 (RA-/Shh-), and (3) The Control (non-treated human UCB-MSCs). The miRNA sequencing revealed a significant up-regulation in the expression of miR-9-5p and miR-324-5p at the early stage of differentiation. But the expressions of miR-137 and let-7b were down-regulated at the next stage of the differentiation in Test 2. They were all responsible for neuron and motor neuron differentiation and suppression of proliferation in neural progenitor cells. Also, we could find some novel miRNAs involved in cholinergic, JAK-STAT, Hedgehog, and MAPK signaling pathways during differentiation.21. Despite many studies that suggested the inhibition of miRNA biogenesis results in inappropriate neuro-regulation, the biology of miRNAs during neuronal differentiation has not properly been understood. Indeed, there is little knowledge regarding the role of miRNAs in the process of motor neuron development. The previous studies have failed to capture the dynamic profile of miRNA expression during neurogenesis22. Therefore, this study evaluates the miRNA profile of differentiating hUCB-MSCs during differentiation into motor neuron-like cells. This procedure was accomplished by applying RA and Shh and developing a two-step protocol. At the first step of the induction protocol, the RA and Shh were employed synergistically to mimic the developmental pattern of the neural tube through the rostrocaudal (by RA) and dorsoventral (by Shh) axes. Then, RA and Shh were removed, and the cells were incubated with a neurotrophic factor, naming BDNF. In the previous study, the authors assessed the differentiation potential of human chorion-derived mesenchymal stem cells into motor neuron-like cells in two- and three-dimensional culture systems. Similarly, it is confirmed that chorion-derived mesenchymal stem cells conveniently access various types of cells, which can differentiate into motor neuron-like cells in the presence of RA and Shh in a two-dimensional culture system and on an electro spun gelatin scaffold11.The miRNAs have been known to be the most remarkable regulatory molecule involved in neurogenesis23. In 2016, Sim et al.24 studied the brain-enriched miRNA , which regulates synaptic plasticity and memory. They demonstrated that miR-9-3p and miR-9-5p are involved in the normal development of the central nervous system. This procedure was carried out by striking a balance between differentiation and self-renewal of neural stem cells. The miR-9 affects the TLX expression in the neural stem cells25. The TLX expression is constrained during the differentiation of neural stem cells. But the miR-9 expression increases at the same time. Also, the miR-9 controls the differentiation of early motor neurons into mature motor neurons by regulating the expression of the Onecut transcription factor. Moreover, the Onecut supports the expression of Islet-1 and the lateral motor column LMCms. The interaction of Islet-1 and another transcription factor  supports the formation of the lateral motor column28.In the present study, the hsa-miR-9-5p expression could be detected after induction with RA and Shh in Test 1 group. However, the expression of this miRNA significantly dropped in Test 2 group. The miR-9 was known to be a highly conserved miRNA in the central nervous system. This miRNA regulates the normal development of the brain29 demonstrated that the development of the Lhx1p lateral motor column depends on the RA secreted from lateral motor column LMCms. Also, Otaegi et al.27 confirmed that regulatory networks are currently unknown within the downstream of RA and the upstream of Islet-1/Lhx1. The FoxP1 is another transcription factor involved in preganglionic and lateral motor neurons. The miR-9 controls motor neuron formation by regulating the level of FoxP1 expression.In 2017, Schaller et al.28 expressed that miR-let7b reduces the self-renewal properties of the neural progenitor cells in the brain of old adults. Indeed, miR-let7b controls the developmental fate of the neural progenitor cells into neurons or glial cells. It is performed by regulating the expression of HMGA2 and HES5 in the notch signaling pathway.The results indicated a significant down-regulation of miR-let7b in Test 2 group compared to the Control group. The down-regulation of miR-let7b supports the proliferation of neural stem cells. This miRNA targets TLX (a nuclear receptor and its effector cyclinD1) to control the proliferation and differentiation of neural stem cells. In 2014, Patterson et al.30.In the Test 1 group, the up-regulation of miR-324-5p could be identified in the presence of Shh as an inducing factor. This miRNA targets Gli-1 as a member of the Shh signaling pathway31. Also, it targets H3K27 in EZH2 methyltransferase to control the proliferation and differentiation of neural progenitor cells in adults. It seems that miR-137 is involved in regulating the maturation of neurogenesis in the hippocampus. Also, it was managed to detect the expression of miR-132 in Test 2. This miRNA is expressed in neurons, but it was not in astrocytes or neural progenitor cells. Similar to miR-137, miR-132 is prevalent in dendrites. Unlike miR-137, miR-132 reduces the volume of the spine32.The findings showed a significant down-regulation for the miR-137 expression in the Test 2 group by removing the RA and Shh. According to previous reports, miR-137 regulates the maturation of neurons. The down-regulation of this miRNA supports the morphogenesis of dendrites, cellular phenotypes, and the development of the spinal cord and early neural cells. The miR-137 regulates the ubiquitin ligase expression, called Mind Bomb-1In this study, some novel miRNAs were exclusively detected in one group or shared between other groups. For example, the miRs-3, 8, and 13 were expressed in the Control, Test 1, and Test 2 groups, respectively. A total of eight miRNAs were common only in Test 1 and Test 2 groups.Also, we considered four candidate signaling pathways involved in neurogenesis, including the acetylcholine signaling pathway, Hedgehog signaling pathways, JAK-STAT signaling pathway, and MAPK pathway. After neural induction in Test 1 and Test 2 groups compared to the Control group, the results indicated the expression of miRNAs involved in the acetylcholine pathway. The novel miRNAs related to Shh signaling pathway were up-regulated in Test 1 group. It was confirmed by comparing the achieved results with the Test 2 group. This issue was probably due to the presence of Shh in the induction medium of the Test 1 group.In the JAK-STAT signaling pathway, some novel miRNAs were up-regulated in Test 2 group. It was confirmed by comparing the attained results with the Control and Test 1 groups.In the MAPK pathway, some novel miRNAs were found in test groups. It was confirmed by comparing the outcomes with the non-treated mesenchymal stem cells.This study revealed that CB-MSCs are potent to express the motor neuron-related markers at the mRNA level and the proteins in the presence of Shh and RA. On the other hand, the analysis of the miRNA profile of motor neuron-like cells showed a significant up-regulation in the expression of miR-9, miR-let7b, miR-137, and miR-324-5p, which are involved in the neural differentiation and neuron/motor neuron maturation. The miR-9-5p and miR-324-5p  were up-regulated at the early stage of differentiation. But the expressions of miR-137 and miR-let-7b (considering cell proliferation regulatory roles) were down-regulated at the next stage of differentiation. Moreover, some novel miRNAs related to the Cholinergic, Hedgehog, MAPK, and JAK-STAT signaling pathways were detected in the test groups. However, further studies are still necessary to validate their functions in the mechanisms related to motor neuron differentiation and maturation.Supplementary Information.


Background and objectiveThe ambiguous nature and high infectivity of the coronavirus disease 2019 (COVID-19) have caused soaring morbidity and mortality worldwide. Real-time polymerase chain reaction (RT-PCR) is preferred for detecting COVID-19. However, its poor sensitivity and the emerging use of high-resolution CT (HRCT) scan for disease severity make the use of RT-PCR quite obsolete. In light of this, our study aimed to explore the beneficial role of HRCT and compare the HRCT findings across various patient demographics and parameters.MethodsThis cross-sectional study included 100 patients with clinical suspicion of COVID-19. All patients underwent a chest HRCT scan preceded by RT-PCR testing. We used the CT severity score (CTSS) of the chest to calculate disease severity. Demographical data and results of radiological findings were tabulated and compared across RT-PCR positivity, age, and gender. Independent samples t-test and chi-square test were used to analyze the data.ResultsGlass ground opacity was the most prevalent finding in 99% of the patients, followed by lymph node involvement, consolidation, and crazy-paving pattern. Pleural effusion was observed in only 10% of the patients while pericardial effusion and hiatal hernia were present in 5%. In RT-PCR-positive patients, the posterior basal segment of the lower lobe of the right and left lungs were found to be dominantly involved; however, the upper and middle lobes of the right lung were more commonly involved than the left lung. The mean CTSS was significantly higher in patients aged above 50 years (p<0.001). The mean CTSS of RT-PCR-negative patients was higher than that of RT-PCR-positive patients .ConclusionRT-PCR has a limited role in the diagnosis of COVID-19. The HRCT scan can detect typical COVID-19 findings even in patients with negative RT-PCR results. Moreover, the use of HRCT scan in determining the disease severity and extent of lung damage can lead to a better assessment of critically ill patients. The coronavirus disease 2019 (COVID-19) pandemic has severely paralyzed healthcare systems worldwide . All theFor diagnosing COVID-19 in suspected patients, a myriad of laboratory and radiological diagnostic modalities are available. These modalities include real-time polymerase chain reaction (RT-PCR), serological testing through enzyme link immunosorbent assay (ELISA), chest radiography, and high-resolution CT (HRCT) scan . CurrentThe CT severity score (CTSS) of the chest is used for assessing the disease severity and the extent of lung damage . It inclIn this study, we aim to explore and investigate the role of HRCT scan in COVID-19 while examining the prevalence of various pulmonary and extrapulmonary findings. We further aim to compare these findings among patients with positive and negative RT-PCR results. Lastly, we aim to assess the differences in mean CTSS across ages, genders, and RT-PCR positivity. The findings of our study would lead to the implementation of measures to ensure favorable outcomes in COVID-19 patients in terms of disease identification, stratification, treatment, prognosis, and assessment for severity.This was a retrospective cross-sectional study conducted on 100 patients with clinical suspicion of COVID-19. The study was conducted at the Department of Radiology, Citi Laboratories in Rawalpindi, Pakistan from February to April 2021. The inclusion criteria were as follows: suspected COVID-19 patients with typical symptoms , oxygen saturation below 94%, and COVID-19 suspicion based on initial chest X-ray findings . The exclusion criteria consisted of patients having non-viral pneumonia, tuberculosis, asthma, chronic obstructive pulmonary disease, and pulmonary edema. These patients were excluded based on systemic examination and patient history. Data was collected using a self-designed proforma that included demographic details, RT-PCR results, and chest HRCT scan findings. All the patients underwent HRCT scans according to the standard protocol, and chest CTSS was calculated from HRCT scan findings.The HRCT scan was obtained from a multidetector Toshiba Aquilion CT scanner  with a tube voltage of 120 kbp and a current of 350 mA. The supine position was used to obtain the chest HRCT scan. All lung fields were completely assessed from apices to bases and a consultant radiologist (with experience of 10 years in the field) reported all the findings. Thereafter, chest CTSS was calculated from the HRCT scan findings, and findings in each lung segment were reported. Categorical variables were expressed as frequency and percentages, while continuous variables were described using mean ± standard deviations. Independent samples t-test and chi-square test were used to analyze the data. A p-value of below 0.05 was considered statistically significant. Data were entered and analyzed using SPSS Statistics version 23.0 .Our cohort had a total of 100 patients; the mean age of the patients was 54.0 ± 14.0 years (range: 24-83 years). Of these 100 patients, 55 (55%) patients were male and 45 (45%) were females. In patients with proven RT-PCR positivity, there was a significant association with consolidation  and lobe involvement (p=0.001). Cardiomegaly was more common with patients aged more than 50 years . The comparison of HRCT findings across RT-PCR positivity is shown in Table The mean chest CTSS among the patients was 14.5 ± 5.83. The score showed a difference when stratified according to age above 50 years, gender, and RT-PCR positivity, as shown in Table The posterior basal segments of the lower lungs showed the maximum level of involvement in RT-PCR-positive patients (n=78). This is illustrated in Table Our study showed that the most common features detected even in RT-PCR-negative patients were ground-glass appearance and consolidation. A study from Pakistan has reported similar findings, with ground-glass appearance and consolidation among 88.5% and 52.8% of the patients respectively . In our Our study has shown that the chest HRCT scan can detect typical findings of COVID-19 even in patients with negative RT-PCR results. Non-contrast HRCT scan is now a preferred diagnostic modality and plays a more crucial role in the diagnosis of clinically suspected COVID-19 patients . SimilarPatients suffering from COVID-19 present with a vast array of symptoms ranging from asymptomatic disease to life-threatening cytokine storm . The cytThe major limitation of our study was its retrospective cross-sectional design based on a convenience sampling technique. We recommend that further cohort studies be conducted to delve deeper into the diagnostic role of chest HRCT in COVID-19 so that its superiority over RT-PCR can be conclusively established. There is an urgent need for the implementation of guidelines so that the diagnostic accuracy of COVID-19 can be established in a better way.Patients with RT-PCR-negative results may present with typical COVID-19 features on HRCT scan evaluation. Chest HRCT scan can delineate several pulmonary and extrapulmonary findings that aid in determining the disease severity. Upon HRCT scan evaluation, posterior basal and lateral basal segments of the lower lobe of the right lung were found to be dominantly involved. The comparative findings on HRCT scan and CTSS between positive and negative RT-PCR patients demonstrated non-significant differences. Hence, the use of a chest HRCT scan along with clinical correlations could offer a solution for low RT-PCR sensitivity in the diagnosis of COVID-19 and its severity.


These processes caused significantly different changes of structural and magnetic properties of the initially-formed Fe-Co nanostructures. All of them crystallized and their cores were composed of body-centered cubic Fe-Co phase, whereas their oxide shells comprised of a mixture of CoFe2O4 and Fe3O4 phases. However, the annealing carried out in hydrogen-containing atmosphere caused a decomposition of the initial oxide shell layer, whereas a similar process in argon led to its slight thickening. Moreover, it was found that the cores of thermally-treated Fe0.25Co0.75 nanochains contained the hexagonal closest packed (hcp) Co phase and were covered by the nanosheet-like shell layer in the case of annealing performed in argon. Considering the evolution of magnetic properties induced by structural changes, it was observed that the coercivities of annealed Fe-Co nanochains increased in comparison with their non-annealed counterparts. The saturation magnetization (MS) of the Fe0.25Co0.75 nanomaterial annealed in both atmospheres was higher than that for the non-annealed sample. In turn, the MS of the Fe0.75Co0.25 and Fe0.50Co0.50 nanochains annealed in argon were lower than those recorded for non-annealed samples due to their partial oxidation during thermal processing.Thermal treatment is a post-synthesis treatment that aims to improve the crystallinity and interrelated physical properties of as-prepared materials. This process may also cause some unwanted changes in materials like their oxidation or contamination. In this work, we present the post-synthesis annealing treatments of the amorphous Fe This safety ,9,10.4 as the reducing agent in precipitation method usually leads to the formation of nanoparticles [The usage of NaBHarticles ,11,12,13articles ,15,16,174 often reveal amorphous or nanocrystalline nature mainly due to the incorporation of a very small amount of boron [It is also known that the nanomaterials synthesized with NaBHof boron . This caof boron ,19,20,21of boron ,24,25,26of boron ,17 in twof boron ,28 or caof boron ,30.x1−Cox  Wire-like nanochains were synthetized according to our previous reports describing the MFI co-reduction reaction [2·xH2O; 98%, Carl Roth GmbH, Karlsruhe, Germany) and cobalt(II) chloride hexahydrate  were dissolved in 300 mL of deionized water. These mixtures served as the reaction precursors. In turn, 1.4 g of sodium borohydride  dissolved in 175 mL of deionized water was used as a reducing agent and was dropped to the previously prepared precursor solutions. The process was carried out in the inert argon gas  and also in the average external magnetic field of about 0.05 T induced by two parallel neodymium magnets.The Fereaction ,17. In tThe as-prepared nanomaterials were rinsed three times with ethanol , Gliwice, Poland) and then three times with acetone  in order to clean the as-prepared Fe-Co nanochains. After that, they were dried at 50 °C in a vacuum for 2 h.0.75Co0.25, Fe0.50Co0.50, and Fe0.25Co0.75 nanochains were subjected to a thermal treatment in a tubular furnace in two different atmospheres. One of them was a mixture of 80% nitrogen and 20% hydrogen , whereas the second one was argon . In general, the samples were heated from room temperature to 400 °C with heating rate of 10 °C per minute. Then, they were held at this temperature for 0.5 h. After that, they were gently cooled down to room temperature. At this point, it should also be mentioned that the Fe-Co nanochains were heated at 400 °C in order to omit their sintering. Moreover, the thermally-treated samples in hydrogen-containing atmosphere were assigned as Fe0.75Co0.25 H2, Fe0.50Co0.50 H2, and Fe0.25Co0.75 H2 whereas the annealed chains in argon atmosphere were assigned as Fe0.75Co0.25 Ar, Fe0.50Co0.50 Ar, and Fe0.25Co0.75 Ar.The as-prepared FeThe morphologies and structural properties of as-prepared and thermally treated Fe-Co nanochains were determined with a Zeiss Crossbeam 350 scanning electron microscope  equipped with an EDAX Elite Plus energy dispersive X-ray spectrometer , a JEOL—JEM 1011 transmission electron microscope , a Philips X’Pert Diffractometer  equipped with a Cu X-ray lamp, and a POLON transmission Mössbauer spectrometer .The SEM and TEM measurements were carried out at 5 and 80 kV of accelerating voltage, respectively. Each Fe-Co sample for the SEM investigations was glued to a carbon-type covering an alumina holder. In parallel to SEM imaging, the chemical compositions of as-prepared Fe-Co samples were determined using a EDS technique. The areal EDS data was collected mostly from the nanochains. However, some unwanted signal coming from the conductive carbon-type was also recorded during the EDS measurements mainly due to a thin layer of glued samples. In order to collect the TEM images, the sample was suspended in acetone ), sonicated for 2 min, and then dropped onto a nickel TEM grid coated with holey carbon films (Electron Microscopy Sciences) and air-dried. At this point, it should be underlined that all samples were prepared according to the same above-mentioned procedures.The X-ray diffraction data was collected using a Cu X-ray lamp (λ = 1.5406 Å). The Fe-Co powders were placed on a single crystalline silicon holder. The patterns were collected at room temperature in scattering angle (2θ) range of 30–90° with a step of 0.05° and a scan rate of 0.2 °/min.57Co(Rh) gamma radiation source and a conventional spectrometer operating in vertical geometry. The source of γ-radiation was additionally placed on a vibrator head moving longitudinally to the gamma beam with a speed varying linearly with time. The collected Mössbauer spectra were analyzed using the specialized NORMOS program  applying a minimization procedure and the method of least squares with additional constraints. Relative intensity of the sub-spectra was estimated on the basis of the surface area under the line of the given component. The determined isomer shift values were referred to the signal of pure iron measured at room temperature.The TMS measurements were performed at room temperature using a S) values were determined by fitting to the high-field part of the measured hysteresis loops to the following Equation (1):The magnetic properties were determined at room temperature recording the magnetic hysteresis loops of the as-prepared and thermally-treated Fe-Co materials with a Quantum Design Physical Property Measurement System equipped with a vibrating sample magnetometer (VSM) option. At this point, it should be added that the saturation magnetization  and CoFe2O4 (JCPDS card no. 22-1086) appears in the diffractograms collected for the Fe0.75Co0.25 and Fe0.50Co0.50 samples annealed in both argon and hydrogen-containing atmosphere. In fact, it is hard to differentiate which oxide dominates in the investigated nanochains because their 2θ positions are very similar. It should be also mentioned that the thermally-treated Fe0.25Co0.75 sample contains hexagonal closest packed (hcp) Co phase (JCPDS card no. 05-0727).The evolution of the structural properties between non-annealed and annealed Fe-Co nanochains was traced using the XRD technique. The collected XRD patterns are shown in 57Fe were carried out in order to identify multiple phases present in the as-prepared and annealed Fe-Co nanochains. Although this method is limited to iron-containing phases, if their content in the samples is substantial—as is the case of the tested samples, it can provide valuable information about their phase composition as well as hyperfine parameters.The transmission Mössbauer investigations based on IS = 0.12–0.17 mm/s), whereas the smaller one covers mainly higher values of HMF (35–50 T) and higher isomer shift (IS = 0.23–0.30 mm/s). Both the components are characterized by values of quadrupole splitting close to zero. The ranges of both distributions were chosen on the basis of the quality of the spectral fit. Relative intensities of the components (p) and their mean HMF field values (<B>) are collected in The room temperature Mössbauer spectra collected for as-prepared Fe-Co nanochains are shown in Undoubtedly, the individual parts of HMF distribution have different origins. The larger part related with component 1 represents atoms situated in the cores of spherical particles composing Fe-Co nanochains, in the form of an amorphous alloy containing mainly iron and cobalt. The amorphous state of the cores is stabilized by remnants of the manufacturing procedure, including boron, carbon, possibly oxygen, and other elements identified in investigations performed by means of EDS technique. The high-field part of HMF distribution associated with component 2 is attributed mainly to the oxide shell composed of iron and/or iron-cobalt oxides in a form of crystallites that are strongly distorted, primarily due to the proximity of both surfaces (inner and outer) of the oxide layer. However, the mean HMF values of this component are clearly smaller than typical of most crystalline Fe and Fe-Co oxides ,32,33,34The central part (component 3) covers 5–8% of the spectrum and is composed of a doublet with a line width of about 0.4 mm/s, quadrupole splitting 1.0–1.1 mm/s and isomer shift 0.45–0.48 mm/s , and a singlet with similar line width and isomer shift 0.17–0.19 mm/s. They are attributed to fine particles of iron-cobalt (a singlet) or iron and iron-cobalt oxides (a doublet), which show superparamagnetic behavior at room temperature, including also oxides in the amorphous state.0.75Co0.25 H2 also comprises a small (6%) component IV in a form of a set of more smeared sextets corresponding to the HMF values of about 16, 24, and 28 T. They might be attributed to (Fe-Co)3C or other Fe-Co-B-C structures [The Mössbauer spectra of the thermally treated Fe-Co nanochains shown in ructures ,41. Howeructures .2O4), which covers the core of nanoparticles comprising the nanochains. Relative intensity of this component is very similar to that of the corresponding non-annealed samples except for the sample containing nominally 25% of iron where the intensity of this component is about half lower. This means that heating in the hydrogen-containing atmosphere did not increase or even slightly reduced the relative share of the oxide layer.In turn, the high-field part of the spectra recorded for samples annealed in hydrogen-containing atmosphere was reproduced by two Zeeman sextets with isomer shift between 0.2 and 0.4 mm/s as well as the HMF values of about 41 and 49 T (component II). Its percentage is 4–7% and it represents the shell of distorted iron-cobalt oxides (mainly CoFe2+. The relative share of the whole non-magnetic component ranges from 17% to 19% (component III).In addition, a singlet with isomer shift of about −0.08 mm/s (7–10%) and a wide doublet with isomer shift 1.6 mm/s and quadrupole splitting 1.4 mm/s were found in the central part of the spectra. The former one can be related to the superparamagnetic, small iron groupings located in the fcc cobalt lattice . The lat2O4 phase [2O4 particles. Because the critical size of such particles at room temperature was about 10 nm [In the spectra collected for the Fe-Co nanochains annealed in argon atmosphere shown in b, similaO4 phase . The subut 10 nm ,43, it cHC) of all annealed Fe-Co nanochains are higher in comparison with their non-annealed counterparts. Nevertheless, their values are comparable for both annealing atmospheres. Furthermore, the saturation magnetization (MS) of the Fe0.25Co0.75 nanochains annealed in both atmospheres is higher than that for the non-annealed sample. In turn, the MS of Fe0.75Co0.25 and Fe0.50Co0.50 chains annealed in argon are lower than those recorded for non-annealed samples.The results of room temperature magnetic analyses performed for all investigated Fe-Co nanostructures are demonstrated in 4 usually reveal amorphous or nanocrystalline nature. In fact, this is confirmed in this work. According to the XRD results, the as-prepared Fex1−Cox  Wire-like nanochains synthetized in the MFI co-precipitation reaction reveal only two very broad peaks whose positions are hardly difficult to assign the well-defined crystallographic planes of bcc Fe-Co phase. Moreover, the Mössbauer spectra reveal a smeared shape that is characteristic for strongly disordered systems. Therefore, the post-synthesis annealing at 400 °C in two relatively inert atmospheres, i.e., a mixture of 80% nitrogen and 20% hydrogen, as well as >99% argon gas, was applied in order to improve their crystallinity as well as to investigate the evolution of their structural and magnetic properties.As stated in the Introduction section, the nanomaterials synthesized with NaBH4 taken to the synthesis or the by-products of the process, for instance, B(OH)3. Also, it cannot be excluded that some of the boron forms the borides with the Fe, Co, or both these elements. On one hand, the EDS technique is not sufficient for the determination of the level of light elements , in particular, in small quantities. On the other hand, it is well known that the presence of boron often causes the amorphization of various inorganic compounds [The experimental results obtained in this work indicate that the nominal structural and magnetic properties of the annealed Fe-Co nanochains depend on two factors. The first one is the chemical composition and structure of as-prepared material, whereas the second is related to the applied atmosphere during the thermal treatment. According to the electron microscopy data, the as-prepared Fe-Co nanochains are very similar in terms of their sizes as well as external and internal structures. They are composed of nanoparticles aligned in the nearly straight chains. Moreover, they exhibit characteristic core-shell structures, in which the thick core is covered by the thin shell layer reaching only about 4 nm. Undoubtedly, the presence of a shell layer is associated with an initial oxidation of investigated materials, which was confirmed by the performed EDS and TMS measurements. Besides that, this phenomenon has been frequently observed for the nanomaterials composed of iron and/or cobalt, which stay in contact with atmospheric air ,25,38,42ompounds . This isompounds ,45, the ompounds ,47, as wompounds ,48.The EDS results measured for the as-prepared Fe-Co nanochains indicate that the purification process leads to the complete removal of chloride ions from the samples. They also confirm that the actual share between Fe and Co atoms in the as-prepared samples corresponds well to the composition of initial ion precursors taken to the MFI synthesis.MS of the Fe0.75Co0.25 and Fe0.50Co0.50 nanochains annealed in argon are lower, whereas their MS in hydrogen-containing atmosphere are higher than those recorded for non-annealed samples. This observation is associated with the partial oxidation during thermal processing in argon. At this point, it should be underlined that the Fe0.25Co0.75 sample thermally-treated in both annealing atmospheres reveals the presence of hcp Co phase, which is much more intense than the signal coming from oxides. Therefore, this additional phase hinders the oxidation of the Fe0.25Co0.75 nanochains. This reflects in their morphology and magnetic properties. Namely, the Fe0.25Co0.75 nanochains are covered by a nanosheet-like shell layer in the case of annealing performed in argon. Also, the MS values recorded in hydrogen-containing atmosphere as well as in argon are higher than that for non-annealed sample due to the presence of hcp Co phase. It is also interesting that the HC of all thermally-treated Fe-Co nanochains are higher in comparison with their non-annealed counterparts. The lowest increase of HC is found for the annealed sample in which the content of cobalt is the lowest. In turn, the highest HC value is recorded for the Fe0.50Co0.50 nanochains. These observations are not entirely clear because the HC usually depends on many factors such as: shape, size, presence of internal impurities and/or stresses, ordering, and distances between neighboring chains [Analyzing the electron microscopy images recorded for the thermally-treated Fe-Co nanochains, one can see that their annealing performed in hydrogen-containing atmosphere does not influence their size. In turn, the process in argon causes (i) an increase of their diameters, (ii) a growth of shell layer, as well as leads to the roughening of their surfaces. Undoubtedly, these observations are related to a partial oxidation of the Fe-Co nanochains. This finding is also confirmed by the XRD, TMS, and magnetic measurements. Considering the XRD and TMS results, it is clear that the ratios of the relative intensities between oxide and alloy phases are much higher for the annealing in argon. Moreover, the presence of oxides is hardly noticeable in the XRD patterns recorded for the Fe-Co nanochains annealed in hydrogen-containing atmosphere. Therefore, it is assumed that a very small content of oxide in these samples might be associated with either crystallization of internal oxides or surface oxidation caused by their storage in atmospheric air. In turn, the magnetic tests indicate that the g chains , etc. ThReassuming, the above discussion of experimental results indicates that the annealing atmosphere is quite an important parameter in the case of amorphous Fe-Co Wire-like nanochains and can affect the final chemical and physical properties of thermally treated nanostructures.x1−Cox  Wire-like nanochains, which were prepared in the magnetic-field-induced synthesis. In general, the two atmospheres were investigated, namely: (i) a mixture of 80% nitrogen and 20% hydrogen, and (ii) argon. Both of them are often recognized as inert atmospheres. Therefore, similar results were expected before performance of the experiments. However, the annealing in argon and hydrogen-containing atmospheres led to significantly different changes of structural and magnetic properties of the as-prepared Fe-Co samples.This work presents how the annealing atmosphere influences the structural and magnetic properties of the amorphous Fe2O4 and Fe3O4 oxides. Moreover, it was found that the thermally-treated Fe0.25Co0.75 nanochains were covered by the nanosheet-like oxide shell, which constituted a better barrier against oxidation than oxide layers observed in the case of the annealed Fe0.75Co0.25 and Fe0.50Co0.50 samples. The annealing performed in hydrogen-containing atmosphere also caused crystallization of the cores of the as-prepared Fe-Co nanochains in the form of bbc Fe-Co phase, but their initial oxide shell layers decompose. It is also worth adding that apart from the bcc Fe-Co phase, the cores of the Fe0.25Co0.75 nanochains thermally-treated in both argon and hydrogen-containing atmosphere included hcp Co phase. According to the magnetic measurements, the HC of annealed Fe-Co nanochains increased in comparison with their non-annealed counterparts. It was also found that the MS of the Fe0.25Co0.75 nanochains annealed in both atmospheres were higher than that for non-annealed sample, whereas the MS of the Fe0.75Co0.25 and Fe0.50Co0.50 samples annealed in argon were lower than those recorded for non-annealed nanochains. This observation is related to their partial oxidation during thermal treatment. Finally, it is worth adding that the obtained results can be considered as an important source of information for the application of thermally-treated Fe-Co nanochains as magnetic and microwave absorbers [The thermal processing in argon caused crystallization of the cores of the as-prepared Fe-Co nanochains in the form of bcc Fe-Co phase. At the same time, their diameters of the annealed samples became wider and their surfaces rougher due to the formation of a mixture of CoFebsorbers ,28 or inbsorbers ,30.


Background and purpose — 32-mm heads are widely used in total hip arthroplasty (THA) in Scandinavia, while the proportion of 36-mm heads is increasing as they are expected to increase THA stability. We investigated whether the use of 36-mm heads in THA after proximal femur fracture (PFF) is associated with a lower risk of revision compared with 32-mm heads.Patients and methods — We included 5,030 patients operated with THA due to PFF with 32- or 36-mm heads from the Nordic Arthroplasty Register Association database. Each patient with a 36-mm head was matched with a patient with a 32-mm head, using propensity score. The patients were operated between 2006 and 2016, with a metal or ceramic head on a polyethylene bearing. Cox proportional hazards models were fitted to estimate the unadjusted and adjusted hazard ratio (HR) with 95% confidence intervals (CI) for revision for any reason and revision due to dislocation for 36-mm heads compared with 32-mm heads.Results — 36-mm heads had an HR of 0.9 (CI 0.7–1.2) for revision for any reason and 0.8 (CI 0.5–1.3) for revision due to dislocation compared with 32-mm heads at a median follow-up of 2.5 years (interquartile range 1–4.4).Interpretation — We were not able to demonstrate any clinically relevant reduction of the risk of THA revision for any reason or due to dislocation when 36-mm heads were used versus 32-mm. Residual confounding due to lack of data on patient comorbidities and body mass index could bias our results. During the past years total hip arthroplasty (THA) has become the preferred treatment option for displaced femoral neck fractures in even younger (55–64 years) patients . Previous studies have shown an increased risk of revision, especially due to dislocation, in patients receiving THA after proximal femur fracture (PFF) compared with patients operated due to primary osteoarthritis (OA) , below the threshold of 0.15 that indicates significant imbalance between groups . Mean and standard deviation was used to describe age. Follow-up time was described with median and interquartile range (IQR). PS matching and survival analysis were performed in R software, Version 3.4.4 . PS was calculated using the function “matchit” setting head size as the dependent variable and age, sex, year of surgery, type of fixation, type of bearing, and surgical approach as exploratory variables. The calliper was set to 0.15. Patients from the 36-mm group were matched to the 32-mm group using the nearest neighbor method at a 1:1 ratio. Unmatched patients were discarded from both groups. Kaplan–Meier survival curves for the whole observation time were drawn for each head size. The follow-up period was censored at 7 years because after that time point the number of patients at risk in the 36-mm group dropped below 100. After the 7th year of follow-up only 1 revision occurred in the 32-mm group and none in the 36-mm. The Mantel–Cox log rank test was used to compare the survival curves. Univariable Cox proportional hazards models were fitted to calculate the hazard ratio (HR) for head size with 95% confidence intervals (CI) for the period 0–7 years. Multivariable Cox proportional hazards models were also fitted adjusting for patient age, sex, year of surgery, and surgical approach. Despite the minimal differences in these 4 covariates between the head size groups, we chose to put them in the models in order to block all presumed backdoor pathways (for variables available in NARA database) that could confound the effect of head size on THA revision . 32-mm hThe study was approved by the Regional Ethical Review Board of Gothenburg on October 26, 2016 (reg. ID 858-16). The manuscript was written according to the STROBE  guidelines. No funding specific to this study has been received. 7 co-authors  declare no conflict of interests relevant to this study. 1 co-author (AE) has given paid presentations for and received institutional support from commercial parties (Zimmer-Biomet and DePuy Synthes) related indirectly to the subject of this study. 1 co-author (MM) has given paid presentation for commercial parties  related indirectly to the subject of this study.log-rank = 0.6). The 7-year survival rate was 92.8% (CI 91.2–94.4) for 32-mm and 93.7% (CI 92.2–95.2) for 36-mm heads  1st-time revisions for any reason had occurred in the 32-mm group and 111 (4.4%) 1st-time revisions in the 36-mm group. The Kaplan–Meier survival, although slightly higher for 36-mm heads, did not differ statistically significantly between the 2 head sizes . The 7-year survival rate for THAs with 32-mm heads was 97.8% (CI 97.0–98.7) and 98.3% (CI 97.6–99.0) for 36-mm heads  revisions in the 32-mm and 33 (1.3%) in the 36-mm group. With endpoint revision due to dislocation the Kaplan–Meier survival did not differ significantly between the 2 groups . A non-posterior approach may include different approaches such as direct anterior, anterolateral, lateral, and transtrochanteric that are not specified in the NARA database. However, no major differences in the risk of THA revision have been reported among these surgical approaches . This seems to be a result of the lower survival of 32 mm THA in the Finnish Register, compared with the other 3 registries . This observation should, however, be viewed cautiously since the Finnish Register contributed with significantly fewer 32-mm THAs than 36-mm .Our study could be insufficiently powered to detect smaller risk differences. PS matching at a 1:1 ratio may have caused a considerable loss of statistical power; however, it was the only matching ratio that allowed an acceptable calliper below 0.2, due to the extent of heterogeneity in the unmatched sample. Moreover, our study has inherent limitations due to the lack of randomization. Unmeasured confounding due to factors unknown to us could have skewed the results. For example, the NARA database does not distinguish among femoral neck fracture, trochanteric, or pathologic PFFs. Most of the trochanteric fractures are treated with internal fixation whereas pathologic fractures might receive a THA. Such cases, although probably very few, might be subjected to increased risk of revision due to poor bone quality. BMI and patient comorbidities that increase the risk of THA revision or dislocation (Peters et al. Our results confirm 2 previous reports on patients with femoral neck fracture who received THA and where head size could not be identified as a risk factor for revision. In the study from the National Joint Registry (Jameson et al. Choosing a head size of 36 instead of 32 mm does not seem to be associated with any clinically important decrease in the risk of revision due to any reason and not even due to dislocation after THA in patients operated because of PFF. As THA revision due to dislocation is a rare complication, larger studies with better control of confounders, such as register-based randomized control trials, are needed to make sufficiently powered and unbiased estimations of differences in revision risks between head sizes.Click here for additional data file.


The lack of an efficient, low-cost sequencing method has long been a significant bottleneck in protein research and applications. In recent years, the nanopore platform has emerged as a fast and inexpensive method for single-molecule nucleic acid sequencing, but attempts to apply it to protein/peptide sequencing have resulted in limited success. Here we report a strategy to control peptide translocation through the MspA nanopore, which could serve as the first step toward strand peptide sequencing. By conjugating the target peptide to a helicase-regulated handle-ssDNA, we achieved a read length of up to 17 amino acids (aa) and demonstrated the feasibility of distinguishing between amino acid residues of different charges or between different phosphorylation sites. Further improvement of resolution may require engineering MspA-M2 to reduce its constriction zone's size and stretch the target peptide inside the nanopore to minimize random thermal motion. We believe that our method in this study can significantly accelerate the development and commercialization of nanopore-based peptide sequencing technologies. A new technique for single molecular peptide sequencing is demonstrated by translocation of ssDNA-conjugated-peptide through MspA nanopore which is regulated by a DNA helicase motor. In fact, similar strategies have previously been employed in the investigation of enzyme dynamics,20 direct microRNA sequencing, and epigenetic analysis.21,22 We selected MspA-M2 (D90N/D91N/D93N/D118R/E139K/D134R) as the model nanopore because its constriction is located at the very bottom of the channel, which could theoretically lead to better read length.23,24 We demonstrated that peptides up to 17 amino acid residues could be threaded through the constriction of MspA-M2. Based on the temporal pattern of the generated ionic current, we were able to differentiate point mutations based on their charges and locations in the backbone, as well as phosphorylation sites. Further improvement could be achieved by shrinking the constriction zone and stretching the peptide chain with an enzyme or applying osmotic flow.25,26 Combined, these strategies may provide a feasible pathway to efficient, low-cost peptide sequencing.Compared to well-studied nucleic acid motors, such as polymerases, helicases, translocases, motor proteins that bind to and transport polypeptides have not been extensively characterized.via click reaction to the 5′-end of a handle-ssDNA (ssDNA) , a member of the Hel308 family. Similar to the Hel308 Mbu and Tga helicases already in use for long-read DNA sequencing,20 MTA-h shows significant salinity tolerance and remains functional even at a salt concentration of 600 mM, making it well suited for strand sequencing . Table S3, and det Table S3, S5†. Tog events . For theg events . These r34 Therefore, the dual-MTA-h system can potentially improve the accuracy of peptide sequencing by repeatedly reading the same strand and analysing the signal consensus.35To further improve the read length, we envisaged a tandem arrangement of two MTA-h units to achieve coordinated ssDNA transport over a longer distance . Because5) from the N-terminus of the 23-aa polypeptide (GGGGSGGGGSSGGGGSSGGSGGC) to an array of neutral/hydrophobic amino acid residues and characterized the nanopore translocation of the resultant conjugate strands  to ascertain whether this would lead to further signal augmentation. As seen in 5DD and S5EE exhibited an even more pronounced hump that now exceeded the aforementioned peak amplitude levels that we observed in S5D and S5E. In addition, the hump in the current of mutant G9E, in which G9 was replaced with glutamate, occurred later than its counterpart in S5E or S5D  phosphorylated at S5 began with a steep rising, followed by an extended, high-rising hump, whose amplitude level exceeded those of S5E and S5D, and was on par with those of S5DD and S5EE (9(pS), the signal hump moved to the middle of the peptide-induced signal region , possibly due to the weakening of the electrical field (Fig. S13A careful examination of the blocking current at the ssDNA-peptide transition zone revealed a dense array of upward spikes for all the peptide mutants we tested, except for S and S5K . The ave and S5K . We reas Fig. S13. These f37,38 Potential solutions include engineering MspA-M2 to improve the spatial discrimination of its constriction zone, and stretching the peptide chain by applying electro-osmotic flow or via an enzymatic approach. These improvements could pave the way for our strategy to eventually achieve high-throughput, low-cost single molecule peptide sequencing, with enormous potential in scientific discovery and clinical applications.Here, we have developed a strategy to regulate peptide translocation through the MspA-M2 nanopore and demonstrated its utility in detecting single amino acid residues. The fact that the constriction of MspA-M2 is located at the very bottom of its pore channel allows a read length of up to 17 amino acid residues. The nanopore system that we established could distinguish between amino acid residues with different charge states, and enabled the analysis of post-translational modifications such as serine phosphorylation. We also demonstrated that by using a dual-helicase system, a single peptide could be repeatedly sequenced, potentially leading to significant improvement in sequencing accuracy. Unfortunately, unlike nanopore-based DNA sequencing, peptide translocation did not generate a clear and reproducible step-like ionic current in this study, which precluded sequential discrimination of individual amino acid residues. This observation might be due to the smoothing effect of thermal motion on the blocking current generated by different amino acid residues in an unstrained peptide. Notably, there have recently been studies that achieved step-like blocking current by applying a similar strategy to peptides with a high percentage of negatively charged amino acid residues, confirming the importance of stretching the peptide inside the nanopore.Further data are accessible upon request.J. B. was responsible for the overall research design. Z. C. and Z. W. conducted all the experiments for the study, with input from Y. X., Z. C., Z. W., J. B. analysed experimental data, with input from X. Z and B. T., Z. C. and Z. W. generated all figures used in this study. J. B., Z. C. and Z. W. prepared the manuscript. All authors discussed the results and contributed to the final manuscript.A patent application has been filed by Tsinghua University, which contains the technologies described in this study, with J. B. and Z. C. listed as the inventors.SC-012-D1SC04342K-s001